MUC5B Promoter Polymorphism and Its Association With

Yusuf et al. The Egyptian Journal of Bronchology (2020) 14:18 The Egyptian Journal https://doi.org/10.1186/s43168-020-00015-0 of Bronchology

RESEARCH Open Access A preliminary study: MUC5B promoter polymorphism and its association with IPF Hoda Ali Abu Yusuf1, Mohamed Galal1, Safy Kaddah1, Marwa el Sharkawy2, Mohamed Shaaban Mousa1 and Heba Moussa1*

Abstract Background: The (T) allele of MUC5B gene is strongly correlated with idiopathic pulmonary fibrosis (IPF) and interstitial lung diseases (ILD) related to autoimmune conditions in Caucasians, but no data is available regarding this polymorphism in the Egyptian patients. Results: This study is an observational cross-sectional study; the percentage of the (T) allele of MUC5B gene promoter in normal Egyptian persons in this study was 20%. This polymorphism is strongly related with risk for development of UIP/IPF in Egyptian patients compared to the other 2 groups (P value < 0.001). The MUC5B polymorphism has no role for developing interstitial lung disease in autoimmune diseases. Conclusions: This study showed the potential role of MUC5B promoter polymorphism in IPF patients. Further multicentric studies are essential to be conducted deploying larger cohorts and different ethnic populations for further evaluation of these polymorphisms correlation. Keywords: MUC5B, Gene polymorphisms, IPF

Background Several studies illustrated the potential risk of cigarette Idiopathic pulmonary fibrosis (IPF) is defined as a smoking and other environmental exposures to the pro- chronic progressive type of idiopathic interstitial pneu- gression of IPF [1, 8, 9]. The risk for IPF is mostly deter- monias (IIPs) of unknown cause, occurring mainly in mined by polygenetic variants [10]. It is supposed that elderly, limited to the lungs, and associated with the his- IPF results from the altered behavior of injured alveolar topathologic and/or radiologic pattern of usual intersti- epithelial cells, which produce growth factors that in- tial pneumonia (UIP) [1]. It is the worst form of duce growth of resident fibroblasts and thus fibrocyte idiopathic interstitial pneumonias (IIPs) with a bad prog- accumulation and epithelial to mesenchymal transition nosis [2, 3]. with development of IPF pathology [11]. The incidence of IPF increases with old age with peaks Therefore, many investigations have been carried out in the 6th decade of age, and it is more common in to determine the genetic predisposition for IPF focusing males than females [4]. Approximately, the median sur- on cytokines, growth factors, and the human leukocyte vival is 3 years from time of diagnosis [5]. However, mul- antigen (HLA) group polymorphisms [12–14]. tiple studies on IPF show substantial differences as Many studies confirmed the presence of strong correl- regards demographic distribution in different world re- ation of the MUC5B polymorphisms with IPF in both gion [6]. Worldwide, the mean prevalence of IPF ranges European Caucasian populations and two wide genomic from 6 to 32 per 100,000 [7]. studies [15–19].The common variant rs35705950 is found in the promoter region of the mucin 5B (MUC5B) gene encoding the mucin 5 subtype B. It is both a gel- * Correspondence: [email protected] forming mucin and a major component of mucus in the 1 Chest Department, Cairo University, Giza, Egypt respiratory tract [20, 21]. Seibold et al. [22] were the first Full list of author information is available at the end of the article

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to use linkage and fine mapping to identify the p PCR amplification of the primers terminus of chromosome 11. Interestingly, any pattern Primers to amplify 976 basepair (bp) in the promotor of T allele either heterozygous or homozygous is at area of MUC5B gene (rs35705950) according to the higher risk for IPF. protocol [23]. PCR was performed using hotstar PCR Up to date there is a limited data about the strong cor- mastermix (GeneDirex, Taiwan). relation between MUC5B variant and IPF among the Middle East population. Therefore our study aimed to Detection of the MUC5B gene by RFLP method detect the presence of the common variant of MUC5B Ten microliters of the reaction was digested in a mixture and its association with IPF. that contained 0.5 μl of the restriction enzyme HaeII (Vivantis Technologies, Malaysia), 2 μl of 10X digestion buffer, and 7.5 μl of PCR product for a volume total of Methods 10 ml. The test was executed for 16 h at 37 °C. The prod- Study design uct was separated on a 1.5% agarose gel containing eth- This study was carried out at a pulmonary medicine de- idium bromide (30 min at 120 V) by electrophoresis. The partment in collaboration with the Clinical Pathology genotypes observed under UV illumination were de- department at Faculty of medicine, Cairo University tected as follows: GG genotype showed two bands of (Kasr Alainy hospital) of 18 months duration started 616 bp and 360 bp, the GT genotype showed three bands from November 2015. (976, 616, 360), and the TT genotype showed one band This study is an observational cross-sectional study. It (360 bp) as shown in Fig. 1. consisted of 3 groups: group I, 42 UIP/IPF patients; group II, 47 other ILD patients; and group III, control Statistical analysis group of 40 healthy persons. Pre-coded data was analyzed through the Statistical All IPF patients were diagnosed according to the offi- Package of Social Science Software program, version 23 cial 2013 ATS/ERS consensus criteria [3]. High- (IBM SPSS Statistics for Windows, Version 23.0. resolution chest tomography (HRCT) showed typical Armonk, NY: IBM Corp.). Data was summarized as manifestations of IPF in all patients [1, 3]. ILD patients mean, standard deviation (SD), median, and range for other than UIP/IPF were diagnosed based on clinical quantitative variables, and frequency and percentage for and radiological features, while diagnosis by VATS qualitative ones. Comparison between groups was per- (video-assisted thoracic surgery) was done for the cases formed using independent sample t test (if parametric with atypical radiological image, (five patients were sub- data) or Mann Whitney test (if non-parametric data) for jected to VATS and were diagnosed to have NSIP). The quantitative variables and Chi square and Fisher’s exact latter group (group II) included interstitial lung diseases test for qualitative ones. P values less than or equal to related to autoimmune conditions and they were 13 0.05 were considered of statistical significance. cases whose HRCT showed UIP pattern and 34 cases whose HRCT showed NSIP pattern. Results All patients have the following data: age, sex, past The demographic data of the 3 groups are summarized medical and smoking history, raising birds, occupational in Table 1. and environmental exposure, physical findings, laboratory data, and high resolution computed tomography (HRCT) scans. Patients who suffered from systemic illness with pulmonary affection (i.e., heart failure, renal failure), pulmonary infections, and malignancy (either primary or metastatic to lung) were excluded from the study. The study was finally accepted by the Committee of ethics at the Faculty of Medicine, Cairo University, and consents were obtained from all enrolled persons in this study.

Specimen collection and DNA extraction DNA extraction was performed from 3 ml of peripheral blood on EDTA for all persons using a GF-1 blood DNA Fig. 1 Gel electrophoriesis for the digested PCR products shows the extraction kit (Vivantis Technologies, Malaysia). GG genotype as 2 bands and the GT genotype as 3 bands Yusuf et al. The Egyptian Journal of Bronchology (2020) 14:18 Page 3 of 6

Table 1 Demographic characteristics of the three groups IPF patients (group I) (n = 42) Other ILD patients (group II) (n = 47) Control subjects (group III) (n = 40) Age, years Range 32–58 20–74 25–63 Mean ± SD 47.8 ± 8.8 43.7 ± 11.1 43.2 ± 13.4 Sex Male 17 (40.5%) 10 (21.3%) 10 (25%) Female 25 (59.5%) 37 (78.7%) 30 (75%) Smoking status Smokers 15 (35.7%) 5 (10.6%) 8 (20%) Non-smokers 27 (64.3%) 42 (89.4%) 32 (80%) History of raising birds Yes 14 (33.3%) 17 (36.2%) No 28 (66.7%) 30 (63.8%)

Data demonstrated that the allele frequency of the ge- (group II) with significant difference between the 2 notypes of MUC5B promoter polymorphism were as fol- groups P value 0.0006. In the IPF patients (group 1), we lows: 21 GT (50%), 16 GG (38.1%), 5 TT (11.9%) among detected that the presence of the allele (T) in smokers the IPF patients (group I), and 15 GT (31.9%), 30 GG was higher in 13 patients, P value = 0.014. In the other 2 (63.8%), and 2 TT (4.3%) among other ILD patients groups, the distribution of the (T) allele in smokers was (group II). The frequency for the minor allele (T) of the present in 2 cases with no statistical significance in the polymorphism was 20% in the control group (group III) T allele as regards the smoking status of the subjects (Table 2). The frequency of the minor allele (T) of the (Table 4). polymorphism was significantly higher among the IPF Our results showed there were 14 subjects raising patients in relation to the other two studied groups (P birds among group I compared to 17 subjects raising value < 0.001) (Table 2). birds among group II with no statistical difference be- Although there was female gender predominance tween the 2 groups (P value = 0.779). In the IPF patients among our patients and all were housewives, there were raising birds (group I), the study demonstrated that the 25 IPF female patients (59.5%) (group I) and 37 other presence of the (T) allele in 4 patients, P value = 0.002. ILD patients (78.7%) (group II). However, there was no In the other ILD patient raising birds (group II), the dis- statistical difference among group I and group II as tribution of the minor-allele (T) was present in 6 cases regards the gender. Also, there was no statistical differ- with no significant difference (Table 5). ence in the mean of age among the 3 studied groups. In the healthy group (group III), there was no statis- Discussion tical significance in the T allele between males and fe- In Egypt, a little attention has been paid to define the males. In IPF patients (group I) and other ILD patients possible genetic liability for IPF. In this study, we (group II), there was also no significance in the T allele searched for the correlation between the (T) allele of the detected between males and females (Table 3). mucin gene subtype B and the risk for ILD. The minor There were 15 smokers among the IPF patients (group allele (T) was detected in 6 individuals as heterozygous I) compared to 5 smokers among the other ILD patients genotype (GT) and in only 2 individuals as homozygous

Table 2 The allele frequency of the genotypes of MUC5B promoter polymorphism among the 3 studied groups IPF patients (group I) (n = 42) Other ILD patients (group II) (n = 47) Control subjects (group III) (n = 40) P value Pairs Genotype TT 5 (11.9%) 2 (4.3%) 2 (5%) 0.003 1*2 = 0.043 GT 21 (50%) 15 (31.9%) 6 (15%) 1*3 = 0.001 GG 16 (38.1%) 30 (63.8%) 32 (80%) 2*3 = 0.184 Genotype 1*2 = 0.015 TT or GT 26 (61.9%) 17 (36.2%) 8 (20%) < 0.001 1*3 < 0.001 GG 16 (38.1%) 30 (63.8%) 32 (80%) 2*3 = 0.097 Yusuf et al. The Egyptian Journal of Bronchology (2020) 14:18 Page 4 of 6

Table 3 The genotypes of MUC5B promoter polymorphism as to the studies in the Caucasian population in Europe regards gender among the 3 studied groups and the USA [15, 17, 22]. Genotype Female gender Male gender P value A Chinese study concluded that the frequency of the IPF patients (group I) (T) allele in normal Chinese persons was approximately GT and TT 13 (50%) 13 (50%) 0.109 0.66%, which was lower than that described in the Cau- casian population and our study. However, the frequen- GG 4 (25%) 12 (75%) cies of the T allele were significantly higher among their Other ILD patients (group II) IPF and ILD patients than healthy Chinese controls, GT and TT 14 (82.4%) 3 (17.6%) 0.647 3.33% and 2.22% respectively [23]. GG 23 (76.7%) 7 (23.3%) In this study, the T-allele of the MUC5B in other ILD Control subjects (group III) patients was not significantly different compared to the GT and TT 6 (75%) 2 (25%) 1.000 control subjects. A finding that suggests the absence of any relation between MUC5B polymorphism and lung GG 24 (75%) 8 (25%) fibrosis in the context of autoimmune conditions, either NSIP or UIP. genotype (TT) among the 40 normal individuals, indicat- It is clear that IPF is higher in older age groups. In our ing that the T-allele polymorphism percentage was ap- study, the mean age of IPF patients was (47.8 ± 8.8 proximately 20% in our sample. Other studies conducted years). Our findings were similarly in line to a study con- on other Caucasian populations demonstrated that the ducted by Kaddah et al., which included 102 IPF patients T-allele percentage was 10.81%, 11.74%, 10%, and 9.1% from Egypt; the mean age of their patients was 50 ± 13 in the French, Italian, UK, and in the US Caucasian years [24]. Also, our results goes with that of an Egyp- population, respectively [17–19]. tian study that was conducted in Upper Egypt and Our results revealed that the minor-allele (T) poly- showed that the mean age of their IPF patients was 44 ± morphism in the IPF patients (group I) was present in 12 years [25]. 21 patients as heterozygous G/T genotype and in 5 pa- In multiple studies, IPF appears to have sex predilec- tients as homozygous T/T genotype, denoting that the tion being more common in men; some suppose that T-allele frequency was approximately 61.9% among the this difference may be related to smoking patterns rather IPF patients. Also, the presence of the allele (T) poly- than sex as a risk for IPF [26, 27]. This is against our re- morphism in the other ILD patients (group II) was de- sults in which most of the IPF patients (group I) were fe- tected in 15 patients as the heterozygous G/T genotype males (59.5%). All of them were non-smokers and and in 2 patients as homozygous T/T genotype, showing housewives. Only 33.3% and 36.2% of the female IPF and that the T-allele polymorphism was approximately 36.1% other ILD patients gave history of raising birds respect- among the other ILD patients. The (T) allele frequency ively. There were 17 male IPF patients (40.5%); 35.7% was significantly higher among the IPF patients (group I) gave history of cigarette smoking compared to 10 male in relation to the other two groups (P value < 0.001). IPF patients (21.3%); 10.6% gave history of cigarette Hence, our data support a strong correlation between smoking. Interestingly, our results matched that of a the T-allele of the mucin gene subtype B and the occur- study conducted by Sherbini et al., which included 134 rence of IPF, in the Egyptian patients which is the same IPF patients from Saudi Arabia; 56% of their patients were females and 44% were males; smoking was more evident as 36% of IPF patients were smokers [28]. Table 4 The genotypes of MUC5B promoter polymorphism as In this study, the frequency of the T allele of MUC5B regards the smoking status among the 3 studied groups among male individuals (25%) was lower than female in- Genotype Smokers Non smokers P value dividuals (75%) in the control group, but it was statisti- P IPF patients (group I) cally insignificant ( = 1.000). Moreover, we investigated the possibility of the affection of a function of MUC5B GT and TT 13 (50%) 13 (50%) 0.014 gene by gender among our IPF patients. The results did GG 2 (12.5%) 14 (87.5) not detect any significance in the T allele percentage be- Other ILD patients (group II) tween both female (82.4%) and male ILD (17.6%) pa- GT and TT 2 (11.8%) 15 (88.2%) 0.850 tients and their normal pair controls as well as between GG 3 (10%) 27 (90%) female (50%) and male (50%) IPF patients and matched Control subjects (group III) controls. Borie et al. reported that the T allele risk was not affected by sex in Caucasians [17]. They considered GT and TT 2 (25%) 6 (75%) 0.650 that the mucin 5B subtype could have an independent ef- GG 6 (18.8%) 26 (81.3%) fect on the liability to IPF. However, in the Chinese study, Yusuf et al. The Egyptian Journal of Bronchology (2020) 14:18 Page 5 of 6

Table 5 The genotypes of MUC5B promoter polymorphism as regards the history of raising birds among the IPF patients and the other ILD patients groups Genotype Raising birds subjects Non raising birds subjects P value IPF patients (group I) GT and TT 4 (15.4%) 22 (84.6%) 0.002 GG 10 (62.5%) 6 (37.5%) Other ILD patients (group II) GT and TT 6 (35.3%) 11 (64.7%) 0.925 GG 11 (36.7%) 19 (63.3%) the (T) allele percent was considerably lower in the Chin- despite there were 16 IPF female patients with GG ese population and significantly differed between Chinese phenotype for the MUC5B, the fact that 10 (62.5%) of males and females. The T allele was significantly higher those patients were raising birds may had an influence among male IPF patients compared with the correspond- and played an essential role for development of IPF in ing healthy controls (3.75% versus 0.37%, P = 0.001), de- those patients. The environmental risk factors as raising noting the possible correlation between T allele of the birds may be a potential cause for development of IPF mucin gene subtype B and IPF [23]. even with GG phenotype for the MUC5B subjects. How- Detection of risk factors for IPF is a critical issue in ever, multicentric researches are required to focus on order to take steps towards preventive strategies, early the possibility of relation between the mucin 5B gene diagnosis, and novel therapies. Smoking is one of risk and the bird-raising exposure in the pathogenesis of IPF. factors for IPF. Additionally, smoking appears as a co- In this study, the mucin gene subtype B polymorphism factor for increasing MUC5B expression. Smoking could was not associated with disease severity as evidenced by augment the level of MUC5B expressed by alveolar mac- FVC values and oxygen saturation values of our patients rophages at the long term [29–34]. Recently, the smok- in both groups. However, in the Chinese study per- ing rates were high among both European males and formed by Jiang et al., the MUC5B is associated with IPF females [35, 36]. Among our IPF patients, 15 males were severity [38]. smokers compared to only 5 males of other ILD group This study has limitations as the relatively small num- with positive statistical significance (P value 0.006). Also, bers in each group and high cost of laboratory kits. Fu- the number of the IPF smoker patients was the highest ture researches in Egypt using larger patient cohorts are compared to the other 2 groups (P value 0.016). None of required to confirm the findings of this study. our female patients were smokers. This highlighted that the minor-allele (T) in male IPF smoker patients was Conclusions P high with value = 0.014. Our results suggest that The (T) allele of the MUC5B in the sample of Egyptian smoking could accelerate the process of IPF in patients population is relatively higher than that in other Cauca- with the mucin gene 5B variant. However, due to the sians. This variant significantly associated with the possi- lack of smoking among our Egyptian female patients, we bility of developing IPF in Egyptian patients, although it failed to detect an association between the MUC5B poly- might not be the essential factor for IPF in Egypt. The morphism and female patients with IPF in this study. MUC5B variant is not a risk for lung fibrosis associated One of the suggested risk factors for IPF is bird raising in autoimmune conditions. in Egypt. A multicenter hospital-based case control study from 2010 to 2011 performed in Egypt found Abbreviations strong occupational and exposure associations as wood- IPF: Idiopathic pulmonary fibrosis; ILD: Interstitial lung diseases; IIPs: Idiopathic interstitial pneumonias; UIP: Usual interstitial pneumonia; working and chemical/petrochemical industry for men HRCT: High-resolution chest tomography; HLA: Human leukocyte antigen; and raising birds and farming for women and risk for NSIP: Nonspecific interstitial pneumonia; VATS: Video-assisted thoracic IPF [37]. Our results showed that there were 14 subjects surgery; MUC5B: Mucin 5B gene; RFLP: Restriction fragment length raising birds among the IPF patients (group I) compared polymorphism to 17 subjects raising birds among the other ILD pa- Acknowledgements tients (group II) with no statistical significance between Not applicable the 2 groups (P value = 0.779). The percentage of the minor allele (T) is 4 (15.4%) among IPF raising birds pa- Authors’ contributions tients (group I) while in the other ILD patient raising HAY, SK, MG, and HM had put the design and frame of the work; MS had collected, analyzed, and interpreted data regarding patients with ILD who birds (group II), the configuration of the minor-allele (T) had done HRCT chest. HM had drafted the work, revised it, and was a major was present in 6 (35.3%) cases. Our results showed that, contributor in writing manuscript. MM was a major contributor in Yusuf et al. The Egyptian Journal of Bronchology (2020) 14:18 Page 6 of 6

interpreting pathological data. HAY, SK, and MG had read and approved final 15. Stock CJ, Sato H, Fonseca C et al (2013) Mucin 5B promoter polymorphism manuscript. is associated with idiopathic pulmonary fibrosis but not with development of lung fibrosis in systemic sclerosis or sarcoidosis. Thorax 68:436–441 Funding 16. Peljto AL, Steele MP, Fingerlin TE et al (2012) The pulmonary fibrosis-associated None MUC5B promoter polymorphism does not influence the development of interstitial pneumonia in systemic sclerosis. Chest 142:1584–1588 17. Borie R, Crestani B, Dieude P et al (2013) The MUC5B variant is associated Availability of data and materials with idiopathic pulmonary fibrosis but not with systemic sclerosis interstitial Not applicable. lung disease in the European Caucasian population. PLoS One 8:e70621 18. Fingerlin TE, Murphy E, Zhang W et al (2013) Genome-wide association Ethics approval and consent to participate study identifies multiple susceptibility loci for pulmonary fibrosis. Nat Genet The referred subjects were asked for informed written consent after fulfilling 45:613–620 the inclusive and exclusive criteria of the study. The study was approved by 19. Noth I, Zhang Y, Ma SF et al (2013) Genetic variants associated with the ethical committee of Faculty of Medicine, Cairo University. IRB was not idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide necessary according to national regulations at time of starting the study. association study. Lancet Respir Med 1:309–317 Committee’s reference number is not available. 20. Thornton DJ, Devine PL, Hanski C et al (1994) Identification of two major populations of mucins in respiratory secretions. Am J Respir Crit Care Med Consent for publication 150:823–832 Consent for publication was taken from all authors. 21. Kirkham S, Kolsum U, Rousseau K et al (2008) MUC5B is the major mucin in the gel phase of sputum in chronic obstructive pulmonary disease. Am J – Competing interests Respir Crit Care Med 178:1033 1039 None 22. Seibold MA, Wise AL, Speer MC et al (2011) A common MUC5B promoter polymorphism and pulmonary fibrosis. N Engl J Med 364:1503–1512 Author details 23. Wang C, Zhuang Y, Guo W et al (2014) Mucin 5B promoter polymorphism is 1Chest Department, Cairo University, Giza, Egypt. 2Clinical Pathology, Cairo associated with susceptibility to interstitial lung diseases in Chinese males. University, Giza, Egypt. PLoS One 9:e104919 24. Kaddah S, Ahmed S (2016) Life style associated diseases in patients with Received: 17 April 2020 Accepted: 15 July 2020 intestinal pulmonary fibrosis. The Egyptian society of chest diseases and tuberculosis 65:127–133 25. Rashad MA, Ibrahim AK (2015) Idiopathic pulmonary fibrosis (IPF) in Upper Egypt, a single center study. The Egyptian society of chest diseases and References tuberculosis 64:915–919 1. Raghu G, Collard HR, Egan JJ et al (2011) ATS/ERS/JRS/ ALAT Committee on 26. Olson A, Swigris J, Lezotte D et al (2007) Mortality from pulmonary fibrosis Idiopathic Pulmonary Fibrosis. An official ATS/ERS/JRS/ALAT statement: increased in United States from 1992-2003. Am J Respir Crit Care Med 176: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and 277–284 Am J Respir Crit Care Med – management. 183:788 824 27. Mannino DM, Etzel RA, Parrish RG (1996) Pulmonary fibrosis deaths in the 2. Raghu G, Weycker D, Edelsberg J et al (2006) Incidence and prevalence of United States, 1979–1991. An analysis of multiple-cause mortality data. Am J – idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 174:810 816 Respir Crit Care Med 153:1548–1552 3. American Thoracic Society/European Respiratory Society (2013) An Official 28. Sherbini N, Feteih M, Wali S et al (2014) Idiopathic pulmonary fibrosis in American Thoracic Society/European Respiratory Society Statement: update Saudi Arabia: demographic, clinical and survival data from two tertiary of the International Multidisciplinary Classification of the Idiopathic hospitals. Ann Thorac Med 9:168–117 – Interstitial. Am J Respir Crit Care Med 188:733 748 29. Plantier L, Crestani B, Wert SE et al (2011) Ectopic respiratory epithelial cell 4. Lai CC, Wang CY, Lu HM et al (2012) Idiopathic pulmonary fibrosis in Taiwan differentiation in bronchiolised distal airspaces in idiopathic pulmonary – – a population-based study. Respir Med 106:1566 1574 fibrosis. Thorax 66:651–657. 41 5. Olson A, Swigris J, Lezotte D et al (2007) Mortality from pulmonary fibrosis 30. O’Donnell RA, Richter A, Ward J et al (2004) Expression of ErbB receptors increased in the United States from 1992 to 2003. Am. J. Respir. Crit. Care and mucins in the airways of long term current smokers. Thorax 59:1032– – Med 176(3):277 284 1040. 42 6. Coultas D, Zumwalt R, Black W et al (1994) The epidemiology of interstitial 31. Leikauf GD, Borchers MT, Prows DR et al (2002) Mucin apoprotein – lung diseases. Am J Respir Crit Care Med 150:967 972 expression in COPD. Chest 121:166S–182S. 43 7. Meltzer E, Noble P (2008) Idiopathic pulmonary fibrosis. Orphanet J Rare Dis 32. Choi WI, Syrkina O, Kwon KY et al (2010) JNK activation is responsible for 3:8 mucus overproduction in smoke inhalation injury. Respir Res 11:172–144 8. Hubbard R, Lewis S, Richards K et al (1996) Occupational to exposure to 33. Ekstrom M, Gustafson T, Boman K, et al. Effects of smoking, gender and metal or wood dust and aetiology of cryptogenic fibrosing alveolitis. Lancet occupational exposure on the risk of severe pulmonary fibrosis: a – 347:284 289 population-based case-control study. BMJ 2014, Open 4: e004018. 9. Baumgartner KB, Samet JM, Stidley CA et al (1997) Cigarette smoking: a risk 34. Sepper R, Prikk K, Metsis M et al (2012) Mucin5B expression by lung alveolar – factor for idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 155:242 macrophages is increased in long-term smokers. J Leukoc Biol 92:319–324 248 35. Gorini G, Carreras G, Allara E et al (2013) Decennial trends of social 10. Zhang X, Jiang J, Chen WJ et al (2012) Genetic characterization of a Chinese differences in smoking habits in Italy: a 30-year update. Cancer Causes family with familial idiopathic pulmonary fibrosis. Chin Med J (Engl) 125: Control 24:1385–1391 – 1945 1951 36. World Health Organization (WHO) (2012) Tobacco control in practice. WHO Lancet 11. King TE, Pardo A, Selman M (2011) Idiopathic pulmonary fibrosis. 378: regional office for Europe, Copenhagen – 1949 1961 37. Awadalla NJ, Hegazy A, Elmetwally RA et al (2012) Occupational and 12. Cui Y, Osorio JC, Risquez C et al (2014) Transforming growth factor-beta1 environmental risk factors for idiopathic pulmonary fibrosis in Egypt: a downregulates vascular endothelial growth factor-D expression in human multicenter case-control study. Int J Occup Environ Med 3:107–116 lung fibroblasts via the Jun NH2-terminal kinase signaling pathway. Mol 38. Jiang H, Hu Y, Shang L et al (2015) Association between MUC5B – Med 20:120 134. 12 polymorphism and susceptibility and severity of idiopathic pulmonary 13. Zhang J, Xu DJ, Xu KF et al (2012) HLA-A and HLA-B gene polymorphism fibrosis. Int J Clin Exp Pathol 8(11):14953–14958 and idiopathic pulmonary fibrosis in a Han Chinese population. Respir Med 106:1456–1462 14. Hutyrova B, Pantelidis P, Drabek J et al (2002) Interleukin-1 gene cluster Publisher’sNote polymorphisms in sarcoidosis and idiopathic pulmonary fibrosis. 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